The depletion or down modulation of antiapoptotic protein Bcl-2 is commonly observed during apoptosis mediated by chemopreventive agent benzyl isothiocyanate (BITC) in human pancreatic cancer cells. However, the molecular mechanism by which BITC can attenuate Bcl-2 expression is not clear. Previous findings in the literature suggest that microRNA family such as microRNA-16 (miR-16) could modulate apoptosis in gastric cancer or chronic lymphocytic leukemia cells by targeting BCL2. MicroRNAs (miRNAs) comprising of 19-25 nucleotides, are highly conserved small non-coding RNAs which can regulate normal gene expression for development, cell proliferation and apoptosis by targeting mRNAs of protein coding genes at the posttranscriptional level. Mature miRNAs are capable of binding the RNA-induced silencing complex (RISC) to align with target mRNAs at their 3'untranslated region (3'UTR). Such interaction leads to translational repression or cleavage of miRNAs and the subsequent down-modulation of the respective genes. On this basis, conceptually novel studies, such as exploring the role of miRNA-16 (if any) in the acquisition of BITC sensitivity of human pancreatic cancer cells, were undertaken in our laboratory. Interestingly, pancreatic cancer cells treated with chemopreventive agent BITC revealed up regulation of miR-16 which might be linked to down modulation of antiapoptotic gene BCL2. Since miRNAs are expected to have multiple cellular targets, we also assessed whether BITC can down regulate another potential target of miR-16 a proangiogenic molecule, FGF- 2 as revealed by bioinformatics. Primarily, we plan to determine whether these molecules play important roles in BITC mediated antitumor effect on pancreatic cancer cells. Under the scope of Specific aim 1, the proposed studies will asses whether forced expression Bcl-2 or FGF-2 (lacking 32-UTR region) can rescue cell death and angiogenesis mediated by BITC. Another goal of this proposal is to assess whether overexpression of miR-16 in pancreatic cancer cells can alter anchorage independent growth /apoptosis/angiogenesis and can exert synergistic effect when employed together with BITC. In this R21 pilot proposal, we also like to explore the following objective: the efficacy of benzylisothiocyanate to interfere with the development of PanIN lesions in vivo. Our investigation will be directed to monitor the magnitude of miRNA expression changes by locked nucleic acid in situ hybridization (LNA-ISH) analysis during the progression and regression of early lesions of pancreatic cancer in the context of tumor microenvironment which contributes to the pathological angiogenic process. The following specific aims are proposed for this exploratory project: Aim 1: To investigate the role of benzyl isothiocyanate modulated miR-16 expression and down regulation of its putative targets in pancreatic cancer cells. Aim 2: To investigate whether ectopically expressed miR-16 miRNA can cooperate with BITC to exert anti tumor effect in heterotopic xenograft models of pancreatic cancer. Aim 3: To evaluate the effect of orally administered BITC on the level of miR-16 during the progression of Pancreatic Intraepithelial Neoplasia in a Conditional KrasG12D mouse model. Since MicroRNAs, are increasingly being accepted as playing a crucial regulatory role in normal and dysfunctional cellular processes, this novel observation implicating microRNAs with the antitumor effect of BITC should provide new insights in regard to the control of pancreatic cancer, one of the deadliest of all malignancies. PUBLIC HEALTH RELEVANCE: The long-term goal of this preclinical research project is to assess the role of small molecular weight RNA (micro RNA) in rendering pancreatic cancer cells sensitive to benzyl isothiocyanate, a constituent of many edible cruciferous vegetables. MicroRNAs play a crucial regulatory role in normal and dysfunctional cellular processes. They represent a class of small, noncoding RNA molecules, which have been shown to be involved in almost every human pathology currently under study. From tumor progression and viral host interactions, to immune response and stem cell fate determination, miRNAs are quickly growing in importance as the "master regulators" in cell cycle processes. Given that microRNAs can have multiple target genes and can concurrently decrease the levels of genes with different biological functions, the exogenous expression or silencing of microRNAs should have profound inhibitory effect on pancreatic tumor growth. This strategy for tackling pancreatic cancer, one of the deadliest malignancies of all, is definitely more beneficial than targeting individual genes.